The objective of this research project is to examine the terminal step in the heme biosynthetic pathway, the insertion of iron into protoporphyrin to form protoheme, using chicken erythrocytes as a source of material. Specifically I intend to solubilize and purify ferrochelatase from chicken erythrocytes using techniques that have proven to be successful with bacterial ferrochelatase and other membrane proteins. Once purified the physical properties of ferrochelatase as well as the role of possible cofactors will be determined. The enzyme will be reconstituted into phospholipid vesicles and the nature of the protein-lipid interaction will be examined. Group specific reagents will be used to probe the roles specific amino acid residue side chains may play in interactions with either of the two substrates, iron and protoporphyrin. These data provide a picture of how ferrochelatase performs its specific function. Although all of the enzymes of this pathway in animals are known and it is now generally accepted that the rate limiting step of the pathway is the first enzyme, alpha-aminolevulinate (ALA) synthase, little is known about the mitochondrial membrane bound, terminal enzymes, protoporphyrinogen oxidase and ferrochelatase. The intent of the proposed research is to isolate and characterize ferrochelatase from normal, healthy cells so that it will then become possible to understand what changes occur in this enzyme in porphyric animals. This work will serve as the cornerstone for future research aimed at defining and understanding the role of protein-protein and protein-lipid interactions among the final three enzymes of the pathway.